Chimeric gene formed by the dna sequences that encode the antigenic determinants of four proteins of l. infantum and protein encoded by said gene, and pharmacuetical composition useful for preventing and/or treating leishmaniosis in animals or humans

ABSTRACT

A chimeric polypeptide encoded by the chimeric gene formed by the DNA sequences that encode the antigenic determinants of four proteins of  L. infantum  is disclosed. The protein obtained, rLiPO-Ct-Q (pPQI) has a molecular mass of 38 kD with an isoelectric point of 7.37. This chimeric polypeptide is useful for preventing and/or treating leishmaniosis in animals or humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appln. No.60/113,825, filed Dec. 23, 1998.

OBJECT OF THE INVENTION

The present specification relates to an application for an InventionPatent, regarding a chimeric gene formed of the DNA sequences thatencode the antigenic determinants of four proteins of L. infantum, andto proteins encoded by said chimeric gene, useful for the prevention ortreatment of leishmaniosis, in particular canine leishmaniosis. Theobvious purpose of this lies in using the gene sequence or the proteinobtained from the chimeric gene for providing pharmaceuticalcompositions for preventing or treating leishmaniosis, in particularcanine leishmaniosis, that can be present in the body of a patient, forinstance as a vaccine or a monoclonal antibody preparation. This patientdoes not have to be a dog but can also be a human being who suffers fromdiseases that involve immuno-depression. To achieve this, a chimericgene will be produced that encodes a protein called PQ consisting of achimeric product originating from an “in vitro” synthesis of a chimericgene constructed “ad hoc”, which contains five of the antigenicdeterminants of four different proteins, The product is configured ashighly sensitive and specific for—for instance—generating a protectiveimmune responds against canine Leishmaniosis, or for preparingantibodies against canine Leishmaniosis.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is of utility within the industry dedicated to themanufacture of pharmaceutical products in general.

2. Description of the Related Art

The parasitic protozoa of the Leishmania genus are the aetiologicalagents that cause Leishmaniosis, a range of diseases that have aworld-wide distribution and that are characterised in that they giverise to a wide variety of clinical symptoms.

The main forms of. Leishmaniosis are zoonotic in nature and humans areconsidered as secondary hosts.

The species denoted L. Infantum, widely distributed throughout manyMediterranean areas is the cause of visceral Leishmaniosis (LV) inhumans and dogs,

In fact, dogs infected with L. infantum are the main animal reserve ofthis parasite, particularly during the long incubation period before theclinical symptoms can be observed.

The epidemiological data indicate that there is a direct correlationbetween the prevalence of canine Leishmaniosis and the transmission ofthe parasite to humans. For this reason, it is crucial to detect thedisease or infection early on in campaigns undertaken to control thespread of the disease.

The parasite is transmitted to the host vertebrate as a flagellatepromastigote, by means of a bite of a fly of the family “Phlebotominae”,and the parasite enter the cells of the mononuclear phages where theydifferentiate and reproduce as amastigotes, within the phago-lisosomalstructure.

The infected cells gather in certain tissues, mainly spleen, liver andlymph nodes. It is estimated that around 15 million people are infected-with Leishmaniosis, and every year in the world 500,000 new clinicalcases appear in the world, mainly in the underdeveloped and developingworld.

In the south-western countries of Europe, Visceral Leishmaniosis (VL),is a zoonotic disease caused by the L. Infantum species, as wasmentioned earlier. Recent data derived from epidemiological studiesindicate that there is an alarming incidence of this infection.

In Italy the reported data for incidence of VL ranges from 14.4% to 37%according to the region.

In Portugal, more particularly in the area around Lisbon, seropositiverates of 8.4% have been found and in the region of the French MaritimeAlps different centers of prevalence have been found that vary between3.2% and 17.1%.

In Spain, the prevalence of Leishmaniosis depends on the zone beingstudied. In Catalonia an average incidence rate of 9.3% has beenobserved although in some hot-spots a prevalence of infected dogs of upto 18% has been found.

On the Island of Mallorca, the incidence rate is 14%, and other ratesthat have been found are: 2.4% in Murcia, 8.8% in Granada, from 10 to15% in Salamanca, 5.25% in the province of Madrid, and 14% in Caceres.

Although the number of cases of VL in humans caused by L. infantum canbe considered relatively low, the high percentage of patients withimmuno-depression that become infected by Leishmania could be related tothe high level of this illness in dogs.

In fact, in the South of Europe, 50% of adults that are infected byLeishmaniosis are also patients infected by the HIV virus. On the otherhand, according to these data of Leishmania-HIV co-infection, it hasbeen estimated that the level of infection (by parasites) can be one ortwo orders of magnitude higher than this figure due to the existence ofa large number of undetected infections.

A common characteristic of the different types of Leishmania infectionis that it induces a strong humoral response in the host. Therefore,diagnostic methods based on serological techniques are currently themost widely used.

It has been described that these antibodies are detected even during theasymptomatic phase of the disease in natural and experimentalinfections.

The sensitivity and specificity of these methods depends on the type,source and purity of the antigen used. In immunological processes thatare currently commercialised, complete promastigotes and preparationsmore or less prepared from these are used as a source of antigen. Thismethod normally leads to cross-reactions with serum from patientssuffering from leprosy, tuberculosis, African tripanosomiasis, Chagasdisease, malaria and other parasitosis.

The sensitivity and specificity of the serologic methods depend on thetype, source and purity of the employed antigen. During the last years agreat number of Leishmania antigens have been characterised, some ofthem can be considered as proteins specific to the parasite.

Among these proteins specific to the parasite, the surface proteaseGP63, the surface glycoprotein gp46 and the lipophosphoglicaneassociated KMP-11 protein deserve a mention.

An additional group of Leishmania antigens are formed of evolutionarilyconserved proteins, such as kinesine, thermally induced proteins, actinand tubulin.

As part of a strategy to develop a specific serological diagnosticsystem for Leishmaniosis canine, a laboratory based project has beenundertaken to identify the antigens of L. infantum, by means of aimmuno-detection search of an expression library for genes of L.infantum using dog serum with active visceral Leishmaniosis.

It has been observed that most of the antigens isolated by this methodbelong to the family of proteins conserved during the course ofevolution. The identification of the B epitopes of these antigensindicate, however, that in all cases the antigenic determinants werelocalised in regions that were not well conserved.

In particular, the acidic ribosomal proteins LiP2a and LiP2b arerecognised by more than 80% of the VL sera.

It has been confirmed that these proteins contain disease specificantigenic determinants, and that the recombinant proteins LiP2a andLiP2b, from which a fragment had been removed, could be used as aspecific instrument able to distinguish between VL and Chages disease.

It has also been shown that the PO ribosomal protein of L. Infantum,very highly conserved on the evolutionary scale, is recognised by a highpercentage of VL dog sera. Furthermore, the antigenic determinants arefound exclusively on the C-terminus of the protein, that is to say, inthe region that has been poorly conserved during the course ofevolution.

It has been observed that in 78% of the VL dog sera, antibodies againstH2A protein are also present, and it has been confirmed that despite thesequence identity in all the H2A proteins among eukaryotic organisms,the humoral response to this protein in VL sera is particularly provokedby determinants specific to the Leishmania protein H2A.

The antigenic determinants recognised by the VL dog sera are found atboth termini of the H2A protein.

The obvious solution to the problem currently encountered in this artwould be to have an invention that would allow the assembly of asynthetic chimeric gene that contained the DNA regions encoding theantigenic determinants specific to the proteins LiP2a, LiP2b, LiPO, andH2A, with a view to constructing a protein rich in antigenicdeterminants.

However, as far as the applicant is aware, there is currently noinvention that contains the characteristics described as ideal, with aview to reaching the desired aim. This aim is the construction of aprotein rich in antigenic determinants, arising from the assembly of achimeric synthetic gene, that contains the DNA regions encoding theantigenic determinants specific to the aforementioned proteins.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a chimeric gene formed bythe DNA sequences that encode antigenic determinants of four proteins ofL.infantum, useful for preventing or treating canine Leishmaniosis.

In a further aspect, the invention relates to a protein encoded by saidchimeric gene, containing one or more of the antigenic determinants offour proteins of L. infantum encoded by the chimeric gene.

The invention further relates to method for preventing and/or treatingcanine Leishmaniosis in a human being or an animal. In this therapeuticmethod, the chimeric gene of the invention or the protein encoded by itcan be used. Also, antibodies against the protein encoded by thechimeric gene of the invention, or a antigenic part thereof such as anepitope, can be used.

In further aspects, the invention relates to pharmaceutical compositionsfor the prevention and/or treatment, in humans and/or animals, ofLeishmaniosis, comprising an active substance derived from or directedagainst the chimeric gene of the invention and/or the protein encoded byit, or parts thereof. The active substance is preferably such that itcan be used in a pharmaceutical composition for the treatment and/orprevention of Leishmaniosis.

In particular, the pharmaceutical composition will be in a form of avaccin, containing the protein encoded by the chimeric gene of theinvention, or one or more parts thereof, containing one or more of theantigenic determinants of the protein encoded by the chimeric gene ofthe invention.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition for the prevention and treatment, human or animal, ofleishmaniasis, formed;

a) by the protein Chimera Q, or a variant of this protein containingmodifications or substitutions of preserved amino acids administered toa subject (human or animal), or

b) by protein Q in isolated form or combined with any physiologicaladjuvant by the intraperitoneal, subcutaneous or intramuscular route.

The invention also relates to a vaccine that is able to stimulate theproduction of antibodies that recognize the Leishmania parasite, formed

a) by the protein Chimera Q or a variant of this protein that differsfrom protein Q in preserved amino acids, administered to a subject(human or animal), or

b) by protein Q in isolated form or combined with a physiologicaladjuvant by the intraperitoneal, subcutaneous or intramuscular route.

Furthermore, the invention relates to a pharmaceutical composition forthe prevention and treatment, human or animal, of leishmaniasis formed

a) by protein Q, or a variant of this protein containing modificationsor substitutions of preserved amino acids, combined with the proteinLiHsp70, complete or fragmented, administered to a subject (human oranimal), or

b) by protein Q in isolated form or combined with a physiologicaladjuvant by the intraperitoneal, subcutaneous or intramuscular route.

In yet another aspect, the invention relates to a pharmaceuticalcomposition for the prevention and treatment, human or animal, ofleishmaniasis, formed

a) by any DNA vector bearing the sequence that codes for the proteinChimera Q, or a variant of that sequence that contains modifications orsubstitutions of nucleotides that code for preserved amino acids,administered to a subject (human or animal), or

b) by a DNA vector that contains protein Q combined with a physiologicaladjuvant, administered by the intraperitoneal, intramuscular orsubcutaneous route.

Another aspect of the invention relates to a pharmaceutical compositionfor the prevention and treatment, human or animal, of leishmaniasis,formed a) by any DNA vector bearing:

1) the sequence that codes for the protein Chimera Q, or a variant ofthat sequence that contains modifications or substitutions ofnucleotides that code for preserved amino acids, and

2) the sequence of nucleotides that codes for the protein LiHsp70 orvariants thereof that differs with respect to preserved amino acids,administered to a subject (human or animal), or

b) by any vector that contains the DNA sequence that codes for protein Qas defined under a) administered with a physiological adjuvant by theintraperitoneal, intramuscular or subcutaneous route.

In a further embodiment, the pharmaceutical composition of the inventioncomprises antibodies directed to the protein encoded by the chimericgene of the invention, or parts thereof.

The pharmaceutical preparations of the invention may further contain allknown adjuvants, solvents, buffers etc. known per se for pharmaceuticalcompositions and/or vaccines.

In a further aspect, the invention relates to a method for the treatmentor prevention of Leishmaniosis, using a pharmaceutical composition or avaccin according to the invention, or a preparation comprisingantibodies directed against the protein encoded by the chimeric gene ofthe invention.

This method will generally comprise administering an active substancedirected against the chimeric gene or the protein to a human being oranimal, such as a dog, in a pharmaceutically active amount.

For the prevention of Leishmaniasis, a vaccin comprising the proteinencoded by the chimeric gene, or encoding one or more parts of saidprotein comprising one or more of the antigenic determinants, will beadministered to a human being to elicit a protective immune response.

Administration of the preparations, antibodies and/or vaccins of theinvention may be carried out in a manner known per se, such as orally,intramuscularly, intravenously, subcutaneously, by (drip)infusion etc.Preferably, the preparation or a vaccin is injected, whereas with anantibody preparation, an infusion can be used.

It should be noted that when herein, reference is made to the chimericgene of the invention, this term also encompasses nucleic acid sequencesthat can hybridize with the sequence mentioned below under moderate orstringent hybridizing conditions.

In this context, heterologous hybridisation conditions can be asfollows: hybridisation in 6×SSC (20×SSC per 1000 ml:175.3 g NaCl, 107.1g sodium citrate.5H₂O, pH 7.0), 0.1% SDS, 0.05% sodium pyrophosphate,5*Denhardt's solution (100×Denhardt's solution per 500 ml:10 gFicoll-400, 10 g polyvinylpyrrolidone, 10 g Bovine Serum Albumin (PentaxFraction V)) and 20 μg/ml denatured herring sperm DNA at 56° C. for18-24 hrs followed by two 30 min. washes in 5×SSC, 0.1% SDS at 56° C.and two 30 min. washes in 2×SSC, 0.1% SDS at 56°

For instance, sequences that can hybridize with the sequence mentionedbelow include mutant DNA sequences which encode proteins with the samebiological function as the protein encoded by the sequence mentionedhereinbelow. Such mutant sequences can comprise one or more nucleotidedeletions, substitutions and/or additions to the sequence mentionedbelow. Preferably, the mutant sequences still have at least 50%, morepreferably at least 70%, even more preferably more than 90% nucleotidehomology with the sequence given hereinbelow.

The term chimeric gene as used herein also encompasses nucleic acidsequences that comprise one or more parts of the sequence mentionedhereinbelow. Preferably, such sequences comprise at least 0%, morepreferably at least 30%, more preferably at least 50% of the nucleotidesequence given hereinbelow. Such sequences may comprise a contiguousfragment of the sequence mentioned hereinbelow, or two or more fragmentsof the sequence given below that have been combined in and/orincorporated into a single DNA sequence.

It should be noted that when herein, reference is made to a proteinencoded by the chimeric gene of the invention, this term also includesmutant proteins that still essentially have the same biologicalfunction. Such mutant proteins can comprise one or more amimo aciddeletions, substitutions and/or additions compared to the proteinencoded by the sequence mentioned below. Preferably, the mutant proteinsstill have at least 50%, more preferably at least 70%, even morepreferably more than 90% amino acid homology with the sequence givenhereinbelow.

The term protein also encompasses fragments of the protein encoded bythe chimeric gene of the invention. Such fragments preferably still showthe biological activity of the full protein. Preferably, such proteinscomprise at least 30%, more preferably at least 50% of the amino acidsequence of the full protein. Also, two or more fragments of the fullprotein encoded by the chimeric gene of the invention may be combined toform a single protein.

More specifically, the invention relates to a chimeric gene formed bythe DNA sequences that encode antigenic determinants of four proteins ofL. infantum, encoding a protein useful for pharmacological purposes, inparticular for the prevention and/or treatment of Leishmaniosis, inparticular canine Leishmaniosis, and obtaining the final product or theconstruction of the chimeric gene that encodes a polypeptide thatcontains all the selected antigenic determinants, characterised in thatit uses a cloning strategy in which the clone that expresses the proteinrLiPO-Ct-Q is used as an initial vector, and to this vector, by means ofthe use of suitable restriction sites, fragments of DNA are sequentiallyadded that encode the proteins LiP2a-Q, LiP2b-Q, LiH2A-Ct-Q, LiH2A-Nt-Q,and after each step of cloning the correct orientation of each one ofthe inserts is deduced and the size of the expression products, thecomplete deduced sequence of amino acids of the final fusion protein,pPQV expressed in the pMal vector is;

  MBP   IEGRPLATPRSAKKAVRKSGSKSAKCGLIFPVGRVGGMMRRGQYARRIGA 50SGAPRISEFSVKAAAQSGKKRCRLNPRTVMLAARHDDDIGTLLKNVTLSHSGVV 104PNISKAMAKKKGGKKGKATPSAPEFGSSRPMSTKYLAAYALASLSKASPSQAD 157VEAICKAVHIDVDQATLAFVMESVTGRDVATLIAEGAAKMSAMPAASSGAAAGV 211TASAAGDAAPAAAAAKKDEPEEEADDDMGPSRVDPMQYLAAYALVALSGKTPSK 265ADVQALVKAAGVAVDASRVDAVFQEVEGKSFDALVAEGRTKLVGSGSAAPAGAV 319STAGAGAGAVAEAKKEEPEEEEADDDMGPVDLQPAAAAPAAPSAAAKEEPEESD 373 EDDFGMGGLF(SEQ ID NO.3)

The invention also relates to a pharmaceutical composition for theprevention and treatment, in humans or animals, of Leishmaniasis formed:

a—by the protein Chimera Q, or a variant of this protein which containsmodifications or substitutions of conserved amino acids, administered toa subject (human or animal), either

b—in isolated form or together with any physiological adjuvant via theintraperitoneal, subcutaneous or intramuscular routes.

Also, the invention relates to a vaccine capable of stimulating theproduction of antibodies which recognise the Leishmania parasite, formed

a—by the protein Chimera Q or a variant of this protein which differsfrom protein Q in conserved amino acids administered to a subject (humanor animal), either

b—in isolated form or together with any physiological adjuvant via theintraperitoneal, subcutaneous or intramuscular routes.

Another aspect of the invention comprises a pharmaceutical compositionfor the prevention and treatment, in humans or animals, of Leishmaniasisformed:

a—by protein Q. or a variant of this protein which containsmodifications or substitutions of conserved amino acids, bound toprotein LiHsp70, complete or fragmented, administered to a subject(human or animal), either

b—in isolated form or together with any physiological adjuvant via theintraperitoneal, subcutaneous or intramuscular routes.

A further pharmaceutical composition of the invention for the preventionand treatment, in humans or animals, of Leishmaniasis can be formed:

a—by any DNA vector carrying the sequence which encodes the proteinChimera Q, or a variant of this sequence which contains modifications orsubstitutions of nucleotides which code for conserved amino acids,administered to a subject (human or animal), either

b—by the intramuscular or subcutaneous routes.

In yet another aspect, the invention relates to a pharmaceuticalcomposition for the prevention and treatment, in humans or animals, ofLeishmaniasis formed:

a—by any DNA vector carrying 1—the sequence which encodes the proteinChimera Q, or a variant of this sequence which contains modifications orsubstitutions of nucleotides which code for conserved amino acids, and2—the sequence which encodes the protein LiHsp70, or variants of thesame which differ in conserved amino acids, administered to a subject(human or animal), either

b—by the intramuscular, subcutaneous or intramuscular route.

The invention also relates to a nucleotide sequence and to a proteinuseful for pharmacological purposes, in particular for the preventionand/or treatment of Leishmaniosis, in particular canine Leishmaniosis,having the DNA and amino acid sequence as shown below expressed in thevector PQ31. The amino acid sequence contains a fragment from the vector(AA 1-37). The rest of the amino acid sequence is identical to that ofSEQ ID NO 3 except in the MBP moiety and the first seven amino acids (AA1-7):

1                                                        45 ATG AGA GGATCT CAC CAC CAC CAC CAC CAC ACG CAT CCG CAT GCG Met Arg Gly Ser His HisHis His His His Thr Asp Pro His Ala                  5                  10                  1546                                                       90 AGC TCG AACAAC AAC AAC AAT AAC AAT AAC AAC AAC CTC GGG ATC Ser Ser Asn Asn Asn AsnAsn Asn Asn Asn Asn Asn Leu Cly Ile                 20                  25                  3091                                                      135 GAG GGA AGGCCT TTA GCT ACT CCT CGC AGC GCC AAG AAG GCC GTC Glu Gly Arg Pro Leu AlaThr Pro Arg Ser Ala Lys Lys Ala Val                 35                  40                  45136                                                     180 CGC AAG AGCGGC TCC AAG TCC GCG AAA TGT GGT CTG ATC TTC CCG Arg Lys Ser Gly Ser LysSer Ala Lys Cys Gly Leu Ile Phe Pro                 50                  55                  60181                                                     225 GTG GGC CGCGTC GGC GGG ATG ATG CGC CGC GGC CAG TAC GCT CGC Val Gly Arg Val Gly GlyMet Met Arg Arg Gly Gln Tyr Ala Arg                 65                  70                  75226                                                     270 CGC ATC GGTGCC TCT GGC GCC CCC AGG ATT TCA GAA TTC TCC GTG Arg Ile Gly Ala Ser GlyAla Pro Arg Ile Ser Glu Phe Ser Val                 80                  85                  90271                                                     315 AAG GCG GCCGCG CAG AGC GGG AAG AAG CGG TGC CGC CTG AAC CCG Lys Ala Ala Ala Gln SerGly Lys Lys Arg Cys Arg Leu Asn Pro                 95                 100                 105316                                                     360 CGC ACC GTGATG CTG GCC GCG CGC CAC GAC GAC GAC ATC GGC ACG Arg Thr Val Met Leu AlaAla Arg His Asp Asp Asp Ile Gly Thr                110                 115                 120361                                                     405 CTT CTG AAGAAC GTG ACC TTG TCT CAC AGC GGC GTT GTG CCG AAC Leu Leu Lys Asn Val ThrLeu Ser His Ser Gly Val Val Pro Asn                125                 130                 135406                                                     450 ATC AGC AAGGCG ATG GCA AAG AAG AAG GGC GGC AAG AAG GGC AAG Ile Ser Lys Ala Met AlaLys Lys Lys Gly Gly Lys Lys Gly Lys                140                 145                 150391                                                     495 GCG ACA CCGAGC GCG CCC GAA TTC GGA TCC TCT AGA CCC ATG TCC Ala Thr Pro Ser Ala ProGlu Phe Gly Ser Ser Arg Pro Met Ser                155                 160                 165496                                                     540 ACC AAG TACCTC GCC GCG TAC GCT CTG GCC TCC CTG AGC AAG GCG Thr Lys Tyr Leu Ala AlaTyr Ala Leu Ala Ser Leu Ser Lys Ala                170                 175                 180541                                                     585 TCC CCG TCTCAG GCG GAC GTG GAG GCT ATC TGC AAG GCC GTC CAC Ser Pro Ser Gln Ala AspVal Glu Ala Ile Cys Lys Ala Val His                185                 190                 195596                                                     630 ATC GAC GTCGAC CAG GCC ACC CTC GCC TTT GTG ATG GAG AGC GTT Ile Asp Val Asp Gln AlaThr Leu Ala Phe Val Met Glu Ser Val                200                 205                 210641                                                     675 ACG GGA CGCGAC GTG GCC ACC CTG ATC GCG GAG GGC GCC GCG AAG Thr Gly Arg Asp Val AlaThr Leu Ile Ala Glu Gly Ala Ala Lys                215                 220                 225676                                                     720 ATG AGC GCGATG CCG GCG GCC AGC TCT GGT GCC GCT GCT GGC GTC Met Ser Ala Met Pro AlaAla Ser Ser Gly Ala Ala Ala Gly Val                230                 235                 240721                                                     765 ACT GCT TCCGCT GCG GGT GAT GCG GCT CCG GCT GCC GCC GCC GCG Thr Ala Ser Ala Ala GlyAsp Ala Ala Pro Ala Ala Ala Ala Ala                245                 250                 255766                                                     810 AAG AAG GACGAG CCC GAG GAG GAG GCC GAC GAC GAC ATG GGC CCC Lys Lys Asp Glu Pro GluGlu Glu Ala Asp Asp Asp Met Gly Pro                260                 265                 270811                                                     855 TCT AGA GTCGAC CCC ATG CAG TAC CTC GCC GCG TAC GCC CTC GTG Ser Arg Val Asp Pro MetGln Tyr Leu Ala Ala Tyr Ala Leu Val                275                 280                 285856                                                     900 GCG CTG TCTGGC AAG ACG CCG TCG AAG GCG GAC GTT CAG GCT GTC Ala Leu Ser Gly Lys ThrPro Ser Lys Ala Asp Val Gln Ala Val                290                 295                 300901                                                     945 CTG AAG GCCGCC GGC GTT GCC GTG GAT GCC TCC CGC GTG GAT GCC Leu Lys Ala Ala Gly ValAla Val Asp Ala Ser Arg Val Asp Ala                305                 310                 315946                                                     990 GTC TTC CAGGAG GTG GAG GGC AAG AGC TTC GAT GCG CTG GTG GCC Val Phe Gln Glu Val GluGly Lys Ser Phe Asp Ala Leu Val Ala                320                 325                 330991                                                    1035 GAG GGC CGCACG AAG CTG GTG GGC TCT GGC TCT GCC GCT CCT GCT Glu Gly Arg Thr Lys LeuVal Gly Ser Gly Ser Ala Ala Pro Ala                335                 340                 3451036                                                   1080 GGC GCT GTCTCC ACT GCT GGT GCC GGC GCT GGC GCG GTG GCC GAG Gly Ala Val Ser Thr AlaGly Ala Gly Ala Gly Ala Val Ala Glu                350                 355                 3601081                                                   1125 GCG AAG AAGGAG GAG CCC GAG GAG GAG GAG GCC GAT GAT GAC ATG Ala Lys Lys Glu Glu ProGlu Glu Glu Glu Ala Asp Asp Asp Met                365                 370                 3751136                                                   1170 GGC CCC GTCGAC CTG CAG CCC GCC GCT GCC GCG CCG GCC GCC CCT Gly Pro Val Asp Leu GlnPro Ala Ala Ala Ala Pro Ala Ala Pro                380                 385                 3901171                                                   1215 AGC GCC GCTGCC AAG GAG GAG CCG GAG GAG AGC GAC GAG GAC GAC Ser Ala Ala Ala Lys GluGlu Pro Glu Glu Ser Asp Glu Asp Asp                395                 400                 405 TTC GGC ATGGGC GGT CTC TTC TAAGCGACTC GCCATCTCTT      1256 Phe Gly Met Gly Gly LeuPhe                 410     412 1257 AGCCTCCTTG TGGTGCGCTT GAGGTGCTCTCGCTCTGCTT CTCCTTGCAG 1306 1307 TGTTGGCTGA CTCTGGCGGG TATGTGCCGTCGCATTACAC CCACCTCTCC 1356 1357 CACCCCTTTG CCCTACGCGC TCGCATGCGCAATCCGTGAA TCATCGAGGG 1406 1407 AAGTCTCTCT GGGTGGCAGTGGGTAAGCTT                       1436 (SEQ ID NO.1 and SEQ ID NO.2)

or a mutant or fragment thereof that can be used for generating aprotective immune response in a human or animal against Leishmaniosis,and to a pharmaceutical composition for the prevention and treatment, inhumans or animals, of Leishmaniosis, comprising this protein or a mutantor fragment thereof that can be used for generating a protective immuneresponse in a human or animal against Leishmaniosis. This protein isderived from the insertion of gene PQV in the expression vector pQE31.Here said chimeric gene preferably encodes a polypeptide generated witha molecular weight of 38 kD and an isoelectric point of 7.37.

Also, the invention relates to a vaccine capable of stimulating theproduction of antibodies which recognise the Leishmania parasite,comprising the protein mentioned above or a mutant or fragment thereofthat can be used for generating a protective immune response in a humanor animal against. Leishmaniosis.

A further aspect, of the invention encompasses a pharmaceuticalcomposition for the prevention and treatment, in humans or animals, ofLeishmaniasis, comprising antibodies directed against the proteinmentioned above or a mutant or fragment thereof, preferably containingone or more antigenic determinants such as an epitope.

The invention further relates to a method for the prevention ortreatment of Leishmaniosis in a human or animal, comprisingadministering to the human or animal a pharmaceutical composition asdescribed above, or to a method for preventing Leishmaniosis in a humanor animal, comprising administering to the human or animal a vaccine asdescribed above,

The chimeric gene formed of the DNA sequences that encode the antigenicdeterminants of four proteins of L. infantum and protein obtained,useful for preventing and/or treating Leishmaniosis, that the inventionproposes, in its own right constitutes an obvious novelty within itsfield of application, as according to the invention, a syntheticchimeric gene is produced that as it is obtained by assembly, containingthe DNA region encoding the antigenic determinants specific to theproteins LiP2a, LiP2b, LiPO and H2A, thus constructing a protein rich inantigenic determinants. The chimeric gene obtained is expressed inEscherichia coli and the product has been analysed with respect to itsantigenic properties. The results confirm that this chimeric proteinmaintains all the antigenic determinants of the parent proteins and thatit constitutes a relevant pharmaceutically useful element for canine VL,with a sensibility that oscillates between 80% to 93%, and a specificityof between 96% to 100%.

More particularly, the chimeric gene formed by the DNA sequences thatencode the antigenic determinants of four proteins of L. infantum andthe protein encoded by it, useful for the prevention and/or treatment ofcanine Leishmaniosis and protein obtained object of the invention, isproduced by means of the following stages, namely:

Construction of the chimeric gene. Methodology.

Cloning strategy.

Cloning of DNA sequences that encode antigenic determinants of thehistone protein H2A.

Cloning of the sequences that encode rLiP2a-Q and rLiP2b-Q.

Cloning of the sequence rLiPO-Q.

Cloning of the chimeric gene.

Construction of the chimeric gene from the construction of intermediateproducts.

Cloning of epitopes specific to the L. infantum antigens.

Construction of the final product

Construction of the chimeric gene that encodes a polypeptide thatcontains all the selected antigenic determinants.

optionally expression of the sequence thus obtained.

Evaluation of the final product. sera.

Purification of proteins

Electrophoresis of proteins and immuno-analysis.

Measurements by Fast-ELISA

Evaluation of the final product.

Antigenic properties.

Sensitivity and specificity of the chimeric protein CP in the serumdiagnosis of canine VL.

The strategy followed by the cloning of DNA sequences that encode eachone of the selected antigenic determinants is the same in all cases, andin a first step, the sequence of interest is amplified by means of a PCRand the use of specific oligonucleotides that contain targets forrestriction enzymes at the extremes.

For the cloning step, the amplified product is directed by means of theappropriate restriction enzyme and it is inserted in the correspondingrestriction site of the plasmid pUC18.

After sequencing the DNA, the insert is recovered and sub-cloned to thecorresponding restriction site of the modified plasmid denominatedpMAL-c2. The modification is made by inserting a termination codondownstream of the target HindIII in the polylinker of pMal-c2,denominating the resulting plasmid pMAL-c2*.

Regarding the cloning of the DNA sequence that encodes the antigenicdeterminants of the histone protein H2A, it should be pointed out thatthe cDNA of the clone cL71, that encodes the histone H2A of L. infantum,is used as a template for the PCR reactions, and for the DNAamplification, that encodes the N-terminal region of the histone H2A,more exactly the N-terminal region of the histone H2A, more exactlyrLiH2A-Nt-Q, the following oligonucleotides are used: sense5′-CCTTTAGCTACTCCTCGCAGCGCCAAG-3′ (SEQ ID NO:4) (position 84-104 of thesequence cL71); antisense 5′-CCTGGGGGCGCCAGAGGCACCGATGCG-3′ (SEQ IDNO:5) (inverse and complimentary to position 204-224 of the sequencecL71).

The sequences that are included in the oligonucleotides for the cloningand that are not present in the parent sequence cL-71 are marked inboldface type.

The amplified DNA fragment is cloned directly from the restriction siteXmnI of pMAI-c2*.

The fragment is sequenced by means of the initiator #1234 male and theantigenic C-terminal region of histone H2A, in particular rLiH2A-Ct-Q,is amplified with the following oligonucleotides. These are:

Sense, 5′-GAATTCTCCGTAAGGCGGCCGCGCAG-3′ (SEQ ID NO:6) (position 276-296of the sequence cL71).

Antisense, 5′-GAATTCGGGCGCGCTCGGTGTCGCCTTGCC-3′ (SEQ ID NO:7) (inverseand complimentary to the positions 456-476 of the plasmid cL71).

A triplet that encodes proline (indicated as GGG after the underlinedletters) is included in the anti-sense oligonucleotide, the restrictionsite EccRI that is included in both oligonucleotides for cloning isindicated by underlining.

Regarding the cloning of the sequences that encode rLiP2a-Q, it shouldbe pointed out that the regions of interest are amplified by PCR fromcDNAs encoding LiP2a and LiP2b.

The oligonucleotides that are used for constructing the expression cloneLiP2a-Q, are the following.

Sense, 5′-GTCGACCCCATGCAGTACCTCGCCGCGTAC-3′ (SEQ ID NO:8).

Anti-sense, 5′-GTCGACGGGGCCCATGTCATCATCGGCCTC-3′ (SEQ ID NO:9).

It should be pointed out that the SalI restriction sites added to the 5′extremes of the oligonucleotides have been underlined.

When constructing the expression clone LiP2b-Q, the oligonucleotidesused were:

Sense, 5′-TCTAGACCCGCCATGTCGTCGTCTTCCTCGCC-3′ (SEQ ID NO:10).

Anti-sense, TCTAGAGGGGCCATGTCGTCGTCGGCCTC-3′ (SEQ ID NO:11).

At the 5′extremes of the oligonucleotides the restrictions sites areincluded for the enzyme XbaI (underlined), and due to the cloning needs,an additional triplet, encoding a proline residue, is includeddownstream of the restriction site.

Regarding the cloning of the sequence rLiPo-Q, it should be pointed outthat the cloning of the DNA sequence of the C-terminal region of theprotein PO of L. infantum is carried out by amplifying a clone of cDNAcalled L27 and the following oligonucleotides;

Sense, 5′-CTGCAGCCCGCCGCTGCCGCGCCGGCCGCC-3′ (SEQ ID NO:12) (positions1-24 of the L27 cDNA) and the initiator of the pUC18 sequence (#1211),the amplified DNA is directed by the enzymes PstI+HindIII, with laterinsertion into the plasmid pMAL-c2.

The resulting clone is denominated pPQI and it should be noted that therestriction site PstI is included in the nucleotide with sense(underlined sequence) and that the restriction target HindIII is presentin the cDNA L27.

Regarding the cloning of the chimeric gene, it should be pointed outthat the DNA sequences that encode the five antigenic determinants areassembled into a chimeric gene, and this assembly is carried out on theclone pPQI, to which the codifying regions for the antigenic regionsLiP2a-Q are added sequentially in the 3′ direction (naming the resultsof cloning pPQII), LiP2b-Q (clone pPQIII) LiH2a-Ct-Q (clone pPQIV) andLiH2A-Nt-Q (clone pPQV).

Finally, the insert obtained after the SacI+HindIII digestion of thefinal clone pPQV is inserted into the pQE31 expression plasmid, namingthe resulting clone pPQ.

BRIEF DESCRIPTION OF THE DRAWINGS

To complete the description that is being made and with the aim ofaiding the understanding of the characteristics of the invention, thepresent disclosure is accompanied, as an integral part thereof, by a setof plans of illustrative nature that are not limiting, The following isrepresented:

FIG. 1. Corresponds to the expression (A), purification in amylosecolumns (B) and the antigenicity (C: Western blot; D: ELISA) of each oneof the recombinant proteins fused to the maltose binding protein. Thefigure under D also presents the reactivity in ELISA of each one of therecombinant protein relative to the complete protein.

FIG. 2. Graphic representation of the different vectors considered toobtain the chimeric gene object of the invention, from which thepertinent protein destined to carry out an accurate diagnostic onanimals or human beings that show symptoms of Leishmaniosis will beextracted.

FIG. 3. Corresponds to the identification of the protein obtained fromthe chimeric gene fused to the MBP protein, the preparation of which isrepresented in FIG. 2.

FIG. 4. Corresponds to the expression (A), purification in amylosecolumns (B) and the antigenicity (C: Western blot; F: ELISA) of theintermediates and of the final chimeric protein fused to the maltosebinding protein(PQX-PGV). The figure also represents the expression ofthe chimeric protein (D lane 1), purification in Ni-Nt agarose columns(D lane 2) and the antigenicity of the purified protein against a VLserum (E: Western blot) and against a collection of sera (F, PQ inELISA).

FIG. 5. Shows finally a synthesised graphical representation of thereactivity of a wide variety of canine sera, divided into three groups.The first group contain animals with real infection by L. infantum. Thesecond group includes serum obtained from dogs with various clinicalsymptoms but that are not infected with Leishmania, and a third group ismade up of fifteen control sera from healthy dogs, This figuredemonstrates the value of the invention for carrying out serologicaldiagnosis of VL.

PREFERRED EMBODIMENT OF THE INVENTION

The chimeric gene formed from the DNA sequences that encode theantigenic determinants of four proteins of L. infantum, useful for theserological diagnosis of canine Leishmaniosis and the protein obtainedthat is being proposed are constituted from the construction ofintermediate products. In a first instance, cloning of epitopes specificto the antigens of L. infantum is carried out, which is configured onthe basis of earlier studies on the antigenic properties of four proteinantigens of L. infantum (LiP2a, LiPO, LiP2b, LiH2a), which allow theexistence of B epitopes to be defined for these proteins, and which arespecifically recognised by the canine sera of VL.

With a view to improving the antigenic specificity of these antigenswith respect to the proteins of L. infantum, the specific antigenicdeterminants are cloned from these proteins. After deleting certainregions of these proteins these can be recognised by sera from animalsthat are carriers of VL and other different diseases.

By using the specific oligonucleotides and amplification by PCR ofregions specific to the genes LiP2a, LiP2b, PO and H2A, several clonesare constructed that express the recombinant proteins rLiPO-Ct-Q,rLiP2a-Q. rLiP2b-Q, rLiH2A-Ct-Q and rLiH2A-Nt-Q, just has been detailedin the description of the invention relating to the methodology, wherethe cloning details are described.

The recombinant proteins used are the following;

rLiPO-Ct-Q, which corresponds to the 30 C-terminal residues of theribovomal protein LiPO.

rLiP2a-Q and rLiP2b-Q, that are derived from the ribosomal proteinsLiP2a and LiP2b respectively.

Two sub-regions of the histone H2A, that correspond to the 46 N-terminusresidues (xLiH2A-Nt-Q), and to the 67 C-terminus residues (residues(xLiH2A-Ct-Q).

Each one of the recombinant proteins fused to the maltose bindingprotein (MAP) is expressed in E. Coli, as represented in FIG. No. 1A,and they were purified by affinity chromatography on a amylose column B.After the process of-purification electrophoresis was carried out on therecombinant proteins (lanes 1 to 5).

With the aim of analysing whether the recombinant proteins wererecognised by VL canine sera, a Western blot was incubated, containingthe recombinant proteins in a mixture of three VL canine sera. Giventhat all these proteins are recognised by the sera it is concluded thatthe antigenic determinants present in the parent proteins are maintainedin the recombinant proteins (C).

The antigenic properties of the recombinant proteins are compared withthe antigenic determinants of the parent antigens by means of a FASTELISA, testing against a collection of 26 VL canine sera, just as isshown in the section of FIG. No. 1D, and the fact that the sera showed asimilar reactivity value, both against the selected antigenic regionsand the corresponding complete proteins, demonstrates that no alterationto the antigenic epitope has occurred during the cloning procedure,

In regard to the construction of the final product, more exactly of thechimeric gene that encodes a polypeptide that contains all the selectedantigenic determinants, it should be pointed out that the cloningstrategy is indicated following FIG. No. 2 section A. The intermediateproducts generated during the process are shown.

A clone that expresses the proteins rLiPO-Ct-Q (pPQI) is used as theinitial vector, and the fragments of DNA that encode the proteinsrLiPO-Ct-Q, rLiP2a-Q, rLiP2b-Q, rLiH2A-Ct-Q and rLiH2A-Nt-Q are addedsequentially using appropriate restriction sites.

After each cloning step, the correct orientation of each one of theinserts is deduced from the size of the expression products, and finallythe complete nucleotide sequence of the final clone pPQV is determinedand the amino acid sequence deduced from the sequence represented inFIG. No. 3.

The polypeptide generated has a molecular mass of 38 kD, with anisoelectric point of 7.37, including spacer sequences encoding proline,underlined in FIG. No. 3. The aim of doing this is to efficientlyseparate the antigenic domains and avoid possible tertiary conformationsthat could interfere with the stability and antigenicity of the finalproduct.

The expression and recovery of each of the intermediate products isshown in FIG. No. 4, boxes A and B. As was expected, after eachaddition, the size of the expression product in the vector pMALgradually increases until reaching a molecular weight of 80 kDa,observing a certain degree of rupture during purification.

The chimeric gene was also cloned in the plasmid pQE, a vector thatallows the expression of proteins with a fragment of 6 histidines at theextreme N-terminus. The resulting clone and the recombinant proteins aredenominated pPQ and PQ respectively.

The level of expression of the protein in bacteria transformed with thepPQ plasmid and the purified proteins are shown in FIG. No. 4, referredto in particular with a D, with the protein PQ, purified by affinitychromatography in denaturising conditions is more stable that therecombinant protein pPQV represented in FIG. No. 4, in box D lane 2.

In order to evaluate the final product a series of materials were used,and obviously some techniques, as is described below.

Sera of VL obtained from dogs of different origins are used. The animalsare evaluated clinically and analytically in the pertinent laboratory,generally in a Department of Parasitology, and all the positive sera areassayed for indirect immuno-fluoregence (IIF).

The presence of amastigotes of the parasites of these animals isconfirmed by direct observation of the popliteal and pleescapular lymphnodes, and a second group of 33 sera of VL originating from otherregions, were given a positive diagnosis in the ELISA against totalprotein extracts of the parasite and/or by IIF.

The sera of dogs affected by different diseases that were not VL areobtained from different origins. Within this group sera from thefollowing infections are found:

Mesocestoides spp.

Dyphylidium caninum

Uncinaria stenocephala

Toxocara canis

Dipetalonema dranunculoides

Demodex canis

Babesia canis

Ehrlichia cannis

Ricketsia ricketsiae.

The rest of the sera were obtained from dogs that exhibited variousclinical symptoms that were not related to any infective process, andthe serum controls were obtained from fifteen carefully controlledhealthy animals.

Purification of the recombinant proteins expressed by the clones pMA1-c2is carried out by affinity chromatography on amylose columns, and thepurification of the recombinant protein expressed by the clone pPQ wasperformed on Xi-NTA resin columns in denaturising conditions (Qiagen).

For analysing the proteins, electrophoresis on 10% polyacrimide gels inthe presence of SDS was carried out under standard conditions.Immunological analysis of the proteins separated by electrophoresis wascarried out on nitrocellulose membranes to which the proteins had beentransferred. The transferred proteins were blocked with dried 5% skimmedmilk in a PBS buffer with 0.5% Tween 20.

The filters were sequentially brought into contact with primary andsecondary anti-serum in blocking solutions and an immuno-conjugatelabelled with peroxidase was used as second antibody, visualizing thespecific binding by means of an ECL system. FIG. 4E shows a Western blotof protein PQ.

The Fast-ELISA was used instead of the classic ELISA, and thesensitisation of the antigen was carried out for 12 hours at roomtemperature.

The plates were sensitised with 100 μl of antigen whose concentration inall cases was 2 μg/ml.

After sensitising the wells the plates were incubated for 1 hour withblocking solution (0.5% powdered skimmed milk dissolved in PBS—0.5%Tween 20 and the sera were diluted three hundred fold in blockingsolution).

The wells were incubated with serum for 2 hours at room temperature, andafter exposure to the antibody the wells were washed with PBS-Tween 20.

Antibodies labelled with peroxidase were used as second antibodies at adilution of 1:2000 and the color of the reaction was developed using thesubstrate ortho-phenylenediamine, measuring the absorption at 450 nm.

In regard to evaluation of the final product, it should be pointed outthat the antigenic properties were determined by means of the pertinentstudy of the reactivity of the VL canine sera against the chimericprotein and against each one of the intermediate products in a “Westernblot” assay. All the intermediate products maintained their antigenicityas well as did the final pPQV product, throughout the whole of thecloning process (FIG. 4C).

It should also be pointed out that the recombinant protein expressed bythe pPQ plasmid was recognised by the VL sera. With a view to analysingwith greater precision the antigenic properties of the chimeric proteinand the intermediate products, an analysis of the reactivity of a widevariety of VL canine sera was performed by means of a fast-ELISA againstthe recombinant proteins, as is shown in the section F of FIG. No. 4. Itcan be highlighted that the sensitivity of the different intermediateproducts of cloning increases after each addition step. It should alsobe pointed out that the protein pQI is recognised by most of the VL seraand the protein PQII equally by most of the sera. This proportion isgreater for the protein PQIII and the proteins PQIV, PQV and PQ arerecognised by practically all the sera.

According to what has been discussed above, the percentage ofrecognition shown by the sera was similar both in the case of assayingthe chimeric proteins PQV and PQ, and of assaying a mixture ofrecombinant proteins rLiPO-Ct-Q, rLiP2a, rLiP2b and rLiH2A. It was seenthat the antigenic properties of each one of the 5 selected antigenicregions are present in the PQ expression product, and therefore thisproduct can be used for diagnosis instead of a mixture of the antigensexpressed individually.

With a view to determining whether the chimeric protein can be used forcanine VL serum diagnosis, and according to the pertinent analysis of awide variety of canine sera against this protein, bearing in mind thataccording to the clinical characteristics of the animals, the caninesera have been classified into three groups. A first group consisted ofsera from dogs with a real L. infantum infection, A second group wascomposed of sera of dogs that had various clinical symptoms withoutbeing infected with Leishmania, including dogs infected with parasitesdifferent to Leishmania, and that could exhibit clinical symptoms thatcould be confused with those observed during Leishmaniosis.

The third group was made up of control sera, originated from healthydogs.

In FIG. 5 the average values of reactivity are shown for each group ofsera, the reactivity of the VL sera reaching an average reactivity valueof 0.8 (S.D.=0.4).

Within this group the reactivity of 12 sera was positive but less than0.35, while the reactivity of 10 sera reaches values of between 0.35 and0.5. It was observed that the reactivity of 23 sera varies between 0.5and 1.0, with 14 sera showing a reactivity greater than 1.0.

The average absorption value of the sera of the second group, that is tosay, the group infected with parasites different to Leishmaniaparasites, is 0.2 (S.D.=0.05) and the reactivity of the control sera,that is to say, the third group, is 0.1 (S.D.=0.003). Only two sera fromgroup 2 showed reactivity between 0.35 and 0.40.

The data presented above indicate that the chimeric protein PQ in theFAST ELISA has a sensitivity of 80% for the VL diagnosis, if the cut-offvalue is defined as the average reactivity value of the sera of group 2plus three S.D.'s (that is to say 0.35).

The sensitivity of the assayed group reaches 93%, if the cut-off valueis defined by the reactivity values of the control group. The protein Qhas a specificity of 96% for VL diagnosis, when the cut-off value isdefined by the aforementioned sera of group 2. 100% specificity in theassay was reached when the reactivity values of healthy dogs wereconsidered.

The process to be used is the following;

1.—The microtitre plates are covered with antibodies by incubated 100 μlof a solution that contains 1 μg/ml of antigen dissolved in a bufferPBS—0.5% Tween 20—5% skimmed milk (Buffer A).

The incubation is performed for 12 hours at room temperature, and thenthe plates are washed three times with the same buffer containing noantigen. The dry antigenated plates could be maintained at roomtemperature.

2.—A first incubation of the wells was carried out with the serum ofanimal at a dilution of 1/200 in buffer A. The incubation lasts for 1hour.

3.—The wells are washed with buffer A, as described in point 1, threetimes with a wash flask.

4.—They are incubated with a second antibody (IgG labelled withperoxide) diluted 1:2000 in buffer A, carrying out the incubation for 1hour.

5.—The wells are washed once again with buffer A three times, as wasindicated in the third section, that is to say with a wash flask.

6.—The reactivity is revealed using the substrate ortho-phenylenediamineand the absorption measured at 450 nm.

The protein used for the diagnosis extracted from the chimeric gene isidentified, and the nucleotide sequence encoding said protein, are asfollows:

1                                                        45 ATG AGA GGATCT CAC CAC CAC CAC CAC CAC ACG GAT CCG CAT GCG Met Arg Gly Ser His HisHis His His His Thr Asp Pro His Ala                  5                  10                  1546                                                       90 AGC TCG AACAAC AAC AAC AAT AAC AAT AAC AAC AAC CTC GGG ATC Ser Ser Asn Asn Asn AsnAsn Asn Asn Asn Asn Asn Leu Gly Ile                 20                  25                  3091                                                      135 GAG GGA AGGCCT TTA GCT ACT CCT CGC AGC GCC AAG AAG GCC GTC Glu Gly Arg Pro Leu AlaThr Pro Arg Ser Ala Lys Lys Ala Val                 35                  40                  45136                                                     180 CGC AAG AGCGGC TCC AAG TCC GCG AAA TGT GGT CTG ATC TTC CCG Arg Lys Ser Gly Ser LysSer Ala Lys Cys Gly Leu Ile Phe Pro                 50                  55                  60181                                                     225 GTG GGC CGCGTC GGC GGG ATG ATG CGC CGC GGC CAG TAC GCT CGC Val Gly Arg Val Gly GlyMet Met Arg Arg Gly Gln Tyr Ala Arg                 65                  70                  75226                                                     270 CGC ATC GGTGCC TCT GGC GCC CCC AGG ATT TCA GAA TTC TCC GTG Arg Ile Gly Ala Ser GlyAla Pro Arg Ile Ser Glu Phe Ser Val                 80                  85                  90271                                                     315 AAG GCG GCCGCG CAG AGC GGG AAG AAG CGG TGC CGC CTG AAC CCG Lys Ala Ala Ala Gln SerGly Lys Lys Arg Cys Arg Leu Asn Pro                 95                 100                 105316                                                     360 CGC ACC GTGATG CTG GCC GCG CGC CAC GAC GAC GAC ATC GGC ACG Arg Thr Val Met Leu AlaAla Arg His Asp Asp Asp Ile Gly Thr                110                 115                 120361                                                     405 CTT CTG AAGAAC GTG ACC TTG TCT CAC AGC GGC GTT GTG CCG AAC Leu Leu Lys Asn Val ThrLeu Ser His Ser Gly Val Val Pro Asn                125                 130                 135406                                                     450 ATC AGC AAGGCG ATG GCA AAG AAG AAG GGC GGC AAG AAG GGC AAG Ile Ser Lys Ala Met AlaLys Lys Lys Gly Gly Lys Lys Gly Lys                140                 145                 150391                                                     495 GCG ACA CCGAGC GCG CCC GAA TTC GGA TCC TCT AGA CCC ATG TCC Ala Thr Pro Ser Ala ProGlu Phe Gly Ser Ser Arg Pro Met Ser                155                 160                 165496                                                     540 ACC AAG TACCTC GCC GCG TAC GCT CTG GCC TCC CTG AGC AAG GCG Thr Lys Tyr Leu Ala AlaTyr Ala Leu Ala Ser Leu Ser Lys Ala                170                 175                 180541                                                     585 TCC CCG TCTCAG GCG GAC GTG GAG GCT ATC TGC AAG GCC GTC CAC Ser Pro Ser Gln Ala AspVal Glu Ala Ile Cys Lys Ala Val His                185                 190                 195596                                                     630 ATC GAC GTCGAC CAG GCC ACC CTC GCC TTT GTG ATG GAG AGC GTT Ile Asp Val Asp Gln AlaThr Leu Ala Phe Val Met Glu Ser Val                200                 205                 210641                                                     675 ACG GGA CGCGAC GTG GCC ACC CTG ATC GCG GAG GGC GCC GCG AAG Thr Gly Arg Asp Val AlaThr Leu Ile Ala Glu Gly Ala Ala Lys                215                 220                 225676                                                     720 ATG AGC GCGATG CCG GCG GCC AGC TCT GGT GCC GCT GCT GGC GTC Met Ser Ala Met Pro AlaAla Ser Ser Gly Ala Ala Ala Gly Val                230                 235                 240721                                                     765 ACT GCT TCCGCT GCG GGT GAT GCG GCT CCG GCT GCC GCC GCC GCG Thr Ala Ser Ala Ala GlyAsp Ala Ala Pro Ala Ala Ala Ala Ala                245                 250                 255766                                                     810 AAG AAG GACGAG CCC GAG GAG GAG GCC GAC GAC GAC ATG GGC CCC Lys Lys Asp Glu Pro GluGlu Glu Ala Asp Asp Asp Met Gly Pro                260                 265                 270811                                                     855 TCT AGA GTCGAC CCC ATG CAG TAC CTC GCC GCG TAC GCC CTC GTG Ser Arg Val Asp Pro MetGln Tyr Leu Ala Ala Tyr Ala Leu Val                275                 280                 285856                                                     900 GCG CTG TCTGGC AAG ACG CCG TCG AAG GCG GAC GTT CAG GCT GTC Ala Leu Ser Gly Lys ThrPro Ser Lys Ala Asp Val Gln Ala Val                290                 295                 300901                                                     945 CTG AAG GCCGCC GGC GTT GCC GTG GAT GCC TCC CGC GTG GAT GCC Leu Lys Ala Ala Gly ValAla Val Asp Ala Ser Arg Val Asp Ala                305                 310                 315946                                                     990 GTC TTC CAGGAG GTG GAG GGC AAG AGC TTC GAT GCG CTG GTG GCC Val Phe Gln Glu Val GluGly Lys Ser Phe Asp Ala Leu Val Ala                320                 325                 330991                                                    1035 GAG GGC CGCACG AAG CTG GTG GGC TCT GGC TCT GCC GCT CCT GCT Glu Gly Arg Thr Lys LeuVal Gly Ser Gly Ser Ala Ala Pro Ala                335                 340                 3451036                                                   1080 GGC GCT GTCTCC ACT GCT GGT GCC GGC GCT GGC GCG GTG GCC GAG Gly Ala Val Ser Thr AlaGly Ala Gly Ala Gly Ala Val Ala Glu                350                 355                 3601081                                                   1125 GCG AAG AAGGAG GAG CCC GAG GAG GAG GAG GCC GAT GAT GAC ATG Ala Lys Lys Glu Glu ProGlu Glu Glu Glu Ala Asp Asp Asp Met                365                 370                 3751136                                                   1170 GGC CCC GTCGAC CTG CAG CCC GCC GCT GCC GCG CCG GCC GCC CCT Gly Pro Val Asp Leu GlnPro Ala Ala Ala Ala Pro Ala Ala Pro                380                 385                 3901171                                                   1215 AGC GCC GCTGCC AAG GAG GAG CCG GAG GAG AGC GAC GAG GAC GAC Ser Ala Ala Ala Lys GluGlu Pro Glu Glu Ser Asp Glu Asp Asp                395                 400                 405 TTC GGC ATGGGC GGT CTC TTC TAAGCGACTC GCCATCTCTT        1256 Phe Gly Met Gly GlyLeu Phe                 410     412 1257 AGCCTCCTTG TGGTGCGCTTGAGGTGCTCT CGCTCTGCTT CTCCTTGCAG 1306 1307 TGTTGGCTGA CTCTGGCGGGTATGTGCCGT CGCATTACAC CCACCTCTCC 1356 1357 CACCCCTTTG CCCTACGCGCTCGCATGCGC AATCCGTGAA TCATCGAGGG 1406 1407 AAGTCTCTCT GGGTGGCAGTGGGTAAGCTT                       1436 (SEQ ID NO.1 and SEQ ID NQ.2)

It is not considered necessary to extend this description in order thatsomeone skilled in the art can understand the scope of the invention andthe advantages that it confers.

The materials, form, size and disposition of the elements aresusceptible to change, provided it does not suppose a change in theessence of the invention.

The terms in which this disclosure has been written should always beconsidered as broad in nature and not limiting.

Vaccine Against Leishmania BACKGROUND OF THE INVENTION

The protozoan parasites of the genus Leishmania are responsible forcausing leishmaniasis, a symptomatically complex disease whichessentially affects men and animals in tropical and subtropical regions.It is estimated that the number of new cases of human visceralleishmaniasis can reach the number of 500,000, there being a minimum ofseveral tens of millions of persons affected. Additionally the number ofcutaneous and mucocutaneous leishmaniasis can be of the order of2.000.000 per year (Modabber., 1990). Although the persons at risk ofcontracting the different types of leishmaniasis can be estimated inabout 350 million, the number of persons with real infection can be muchhigher, due to the fact that there are no clear estimations of the realcases of asymptomatic infections and because of the existence of crypticinfections. In fact, leishmaniasis can be considered within the globalcontext as an infection/ disease of endemic nature, situated between the4^(th) and 5^(th) place in the ranking of parasitic diseases withworld-wide repercussion.

Three main forms of leishmaniasis can be distinguished; cutaneous,mucocutaneous and visceral, the characteristics of which mostly dependupon the localisation of the parasite, the species to which it belongsand the clinical manifestations it produces, The species distributedalong Asia and certain regions in the Mediterranean area bring about thepresence of the cutaneous form, with localised ulceration which, in manycases, heal spontaneously. These manifestations are caused by L. majorand L. tropica. L. aethiopica (Mediterranean, Asia, Africa) also inducesthe cutaneous form of leishmaniasis, although its manifestation is morediffuse. In America, the species L. mexicana produces the cutaneous formwith a generalised localization that does not usually healspontaneously. The mucocutaneous form of the disease in humans is causedby L. brasiliensis and is characterised by the presence of cutaneouslesions in oronasal and pharyngeal regions, bringing about thedestruction of the mucosae. In America, Europe, Africa and Asia, themost frequent form of leishmaniasis is the visceral form, caused by L.chagasi, L. donovani and L. infantum. This form of leishmaniasis ischaracterized by clinical symptoms associated to fever, anaemia and anintense hepato/splenomegalia, which is lethal if it is not treatedsuitably at the right time. In the advanced form of the disease, thehost is incapable of developing an effective immune response. All ofthese forms of leishmaniasis are also detected in canids and somerodents which, in fact, constitute the main reservoirs of the parasite.The health problem generated by L. infantum in the Mediterranean basinis serious because there is a high incidence of the infection/disease indogs, and the vector insect is very extended. It is calculated thatbetween 7% and 20% of all canids are infected by Leishmania, reaching30%, in some areas of Spain where it is endemic. This fact constitutes aserious veterinary problem which additionally increases the risk ofcontagion, fundamentally by immunodepressed persons. In Europe, thereare some 11 million dogs at risk of infection by Leishmania.

L. infantum, like the rest of the species in the genus has a dimorphicbiological cycle. The intermediate hosts are insects of the Psychodidaefamily, genus Phlebotomus. In the Mediterranean area of Europe, it hasbeen demonstrated that the species P. arias and P. perniciosus are themain vectors, although the vectors P. papatosi, P. longicuspis and P.sergenti are also present. When the parasite is ingested by the vectortogether with the blood of a vertebrate host, it places itself in thegut in an extracellular form, it transforms into a promastigote and itdivides. The infective forms migrate towards the pharynx and theproboscis, from where they will be inoculated into a new vertebratehost. The promastigotes are characterized in that they have a flagellumand an elongated shape of some 15 to 20 μm in length, with a roundedposterior end and a sharp anterior end. The nucleus is situated incentral position and the kinetoplast at the anterior end. In culturemedia, the parasites exhibit a certain degree of morphologicalvariability. After the inoculation of the promastigotes in the skin ofthe vertebrate host, the establishment, or not, of the infection dependsessentially upon two factors., the existence of a suitable cellpopulation—macrophages—and other cells of the phagocytic mononuclearsystem, and the ability of the parasite to survive and multiply itselfin the interior of these cells.

The first step in the penetration of Leishmania into macrophages is theapproximation and adherence to the plasma membrane of the target cell.In vitro studies seem to indicate that there is no direct chemotacticattraction of the promastigotes over the macrophages. Within theenvironment of tissues, free promastigotes activate complement by thealternative pathway, bringing about the formation of a concentrationgradient of fraction C5a, which attracts macrophages and otherinflammatory cells towards the site of inoculation. Once thepromastigote is within a parasitophorous vacuole, the lysosomes fuse toit forming a phagolysosome. In infections caused by othermicro-organisms, the phagolysosome is the organelle responsible for thelysic and elimination by means of several mechanisms such as theproduction of toxic oxygen radicals, by an oxidative metabolic process,the action of hydrolytic lysosomal enzymes, cationic proteins and lowpH. The survival of the parasite in the phagolysosome is a function ofits ability to resist and avoid said mechanisms.

The existence of an immune response against parasitisation by Leishmaniawhich is both humoral and cellular was discovered from the first momentsin which the disease was studied, and has been revised in numerousoccasions. The type of humoral response depends upon the form ofleishmaniasis. In the cutaneous form, the humoral response is very weak,whereas in the visceral type a high antibody response is observed. Inthe cutaneous affections there is a remarkable cell-mediated response,detectable both in vivo by means of delayed type hypersensitivity tests(DTH), as well as in vitro by means of lymphoblastic transformationtests and macrophage migration inhibition tests. In these cases thetitre of serum antibodies is normally low and directly related to theseriousness of the process. Once the amastigotes are within themacrophages, the resolution of the infection depends essentially uponthe cell-mediated immune mechanisms. The cellular response is determinedby the joint action of macrophages, B cells, several sub-populations ofT-cells, and the different lymphokines secreted by all of them. Theparasitised macrophage processes the Leishmania antigens and expressesthem on its surface by a process mediated by the class II MajorHistocompatibility Complex (MHC-II). Additionally, the macrophagesecretes IL-1, which acts as a second activating signal for theT-lymphocyte. In humans, visceral leishmaniasis or kala-azar ischaracterised by a weakened or absent cellular response, detectable bothby the absence of delayed hypersensitivity (DTH) and by cellproliferation methods. Absence of proliferation of T-cells is detectedeven in the presence of mitogens such as concanavelin A orphytohaemagglutinin and inhibition in the production of IL-2 bystimulated T-cells.

In mice, the population of T-lymphocytes (CD4 phenotype) isheterogeneous and can be divided into at least two sub-populationsaccording to the lymphokines they produce. These cells are essential inthe development of protective immunity against cutaneous leishmaniasis(sub-population Th-1) and are at the same time involved in thesuppression of the protective immune response (sub-population Th-2), Tcells induced in resistant mice C57BL/6 or cured BALB/c mice arepredominantly of the Th-1 type, whereas the cells in uncured BALB/c miceare of the Th-2 type. In general, the lymphokines secreted by thesecells favour the development of the cell line which produces them andhas an antagonic effect on the development of the other sub-population.Thus, IL-4 and IL-10 produced by Th-2 cells contribute to theprogression of the infection, favouring the development of this line.

Additionally, they can act directly on the macrophage, not permittingits activation. However, in mice susceptible of infection by Leishmania,deficient in the game which encodes IL-4, contradictory results havebeen obtained. In some cases it has been observed that the absence ofthis cytokine redirects the response and increases the resistance to theinfection whereas in others no differences are observed in the level ofinfection of the mice. In genetically resistant mice it has beenobserved that the absence of expression of the genes of IFN-γ or itsreceptor, CD40 or the ligand of CD40, increases the is susceptibility tothe infection. The interaction of CD40 and its ligand is necessary forthe production of the cytokine IFN-γ necessary to direct the responsetowards Th-1. Mice with a resistant genetic base, which develop a Th-1type response, become susceptible if they are deficient in theexpression of IL-12., developing a Th-2 type response. Recent datasuggests that the production of IL-12 is important to direct theresponse towards Th-1, and that the absence of IL-4 can avoid the Th-2response.

There are a large number of Leishmania proteins which have an antigeniccharacter essentially characterized by Western Blot methods. In theserum of patients and dogs infected by Leishmania it has been possibleto detect the presence of antibodies against membrane proteins such asgp63, gp 46, PSA and KPM-11. Additionally several antigens ofcytoplasmatic intracellular localisation have been characterised suchas: Hsp70; Hsp83; LIP2a, LIP2b, LILIPO, H2A, H3 and a protein related tokinesin, K39 of L. chagasi. The reactivity of the antibodies, whichrecognise conserved proteins present in the serum of dogs infected by L.infantum is directed towards the least conserved areas of theseproteins. Some of the membrane proteins are very antigenic in naturalinfections. In all the cases of natural infection there is a greatrestriction in the humoral response against the proteins because theantibodies developed during the infective process recognise veryrestricted areas of the same. The levels of IgG normally correspond tothe intensity of the infection reflect the degree and the duration ofantigenic stimulation determined by the parasitic load. A Leishmaniaantigen homologous to the type C kinase receptors (LACK) has recentlybeen described which produces an early response of the Th-2 type. Inmice transgenic for the LACK antigen and with a genetic backgroundsusceptible to the infection, the induction of tolerance to this antigenprotects against the infection by Leishmania major. The anti-Leishmaniaantibodies can destroy the promastigotes in vitro in the presence ofcomplement, promote phagocytosis and induce the adherence of severalparticles to the surface of promastigotes and amastigotes.

The pathologic reaction seems to go in parallel with the density ofparasitised macrophages. In visceral leishmaniasis the macrophagesgenerally distribute themselves in a diffuse manner throughout thedifferent organic tissues. The type of inflammation is is constituted byan important cellular infiltrate with a predominance of lymphocytes andplamatic cells together with hyperplasia of phagocytic cells. Theinflammation brings about alterations in the physiology of the affectedorgans producing serious systemic alterations. In some occasions localgranulomatous inflammation occurs, with appearance of granulomae andmicrogranulomae in the different organs. These granulomae areconstituted by macrophages and histiocytes (affected by parasites ornot) surrounded by plasmatic cells and lymphocytes and, in some cases,by fibroblasts. This inflammatory process is accompanied by an organicreaction with the appearance of characteristic lesions in the affectedorgans. Some authors have found deposits of amyloid substance invirtually the totality of the organs. Some authors have formulated thehypothesis that the damages produced by the disease are not directlyattributable to the aethiologic agent but to the organic reactiontriggered.

Vaccine Against Leishmania

The intense immunity which follows the recovery from cutaneousleishmaniasis has given a great impulse to the development ofprophylactic vaccines against this disease. This immunity is derivedfrom the induction of a T response which has associated to it theproduction of inflammatory cytokines which activate macrophages anddestroy the parasites. The immunological memory in the cases ofinfection is probably maintained by the persistent presence of theparasite in the host in a process known as concomitant immunity.

The first studies regarding vaccination against Leishmania in the decadeof the 40's used live parasites as immunogens, These studies led to theproduction of vaccines which produced significant protection againstsubsequent re-infection. However, the knowledge of the possibility thatlive organisms could produce real infections led to such vaccinationprogrammes not to take place for very long and, on the contrary,interest focused upon vaccines based upon dead parasites. These studiesprovided the first evidence on the possibility of producing effectivevaccines by inoculation of parasites.

The clinical trials which used immunisation with dead Leishmaniapromastigotes also began in the decade of the 40's. These vaccinesyielded remarkable successes as a certain degree of protection wasobserved, which could oscillate between 0 and 82% depending on thepopulation. These vaccines had a smaller effect than live parasitevaccines. The isolation of avirulent clones of L. major which protectmice against infection has also demonstrated that an attenuated vaccineis possible. However, the ignorance of the mutations which lead to theloss of virulence and the risk of production of virulent revertants makethis type of vaccination currently unacceptable.

Recently there has been an important progress towards the identificationof molecularly defined candidate vaccines, such as gp63, gp46/M2, thesurface antigen related to the latter antigen known as PSA-2 and theproteins dp72 and gp70-2, the LACK protein and Kmp11. Specifically, theT epitopes present in protein gp63 have been identified, resulting in-that only some of them are capable of inducing a T response, both ofTh-1 as of Th-2. These antigens induce significant protection in modelanimals when they are administered with adjuvants. Protein PSA-2 ofLeishmania is capable of protecting against infection by L. major byinducing a Th-1 response. To evaluate the mechanism of protection ofthis protein as a vaccine against Leishmania in humans, its ability toinduce T-cell proliferation was studied, in patients which had sufferedleishmaniasis and had recovered from it. It was observed that theprotein is capable of bringing about a strong proliferation of theT-cells of these individuals, but not of controls with no prior historyof infection. The response was of the Th-1 type, as was demonstrated bythe cytokine induction pattern.

Sub-unit vaccines have focused strongly on protein antigens because theyare easy to identify, isolate and clone. However, it is necessary totake into account that not all potential vaccine molecules have to beproteins. In fact, lipophosphoglycan (LPG) plays an essential role inthe establishment of infection. Vaccination with LPG can protect againstinfection with L. major. In spite of the existence of a dogma whichstates that T-cells do not recognise non-proteinaceous antigens, the LPGmolecule seems to be presented to T-cells by Langerhans cells of theskin. Additionally, there is evidence which has demonstrated thatmicrobial glycolipids and other non- proteinaceous molecules canrecognise T-cells when they are presented via the CD-1 route. Althoughthere is no clear evidence that protein KMP11 associated to LPG inducesa protective response, it has been proved that the proteinaceousfraction associated to LPG is capable of inducing a T response andIFN-γ, whereas the LPG fraction without protein is not.

It is normally accepted that sub-unit vaccines although protective, onlyinduce a short term immunity. This problem may not be important inendemic areas, where the individuals can be periodically boosted bycause of natural infections. A major problem in the use of sub-units mayarise from the fact that there may not be a response to a single antigenin a genetically diverse population. A cocktail of antigens containing Band T inducers may overcome this drawback. It has recently beenpublished that an extract of membrane proteins of Leishmania infantum,when injected intraperitoneally, is capable of conferring protectionagainst the virulent promastigote forms of this parasite, and that thisprotection is greater when the proteins are encapsulated in positivelycharged liposomes. Adjuvanticity and protective immunity elicited byLeishmania donovani antigens encapsulated in positively chargedliposomes.

Vaccination with nucleic acids carrying genes which encode Leishmaniaproteins involves the administration of genetic material of the parasiteto the host. This DNA is taken up by the cells and is introduced intothe nucleus where it is transcribed and subsequently translated in thecytoplasm. The advantage of this type of vaccination is that it ispossible to direct the immune response by means of the MHC-I or theMHC-II route. The antigens produced intracellularly are processed in thecell and the peptides generated are presented on the cell surface inassociation with MHC-I molecules. The consequence would be that thesemolecules would give rise to the induction of cytotoxic T-cells. Theantigens produced in an extracellular environment would be speciallytaken up by specialised antigen-presenting cells, processed andpresented on their surface bound to MHC-II molecules, resulting in theinduction and activation of CD4+ cells which secrete cytokines whichregulate the effector mechanisms of other cells of the immune system.

The first DNA vector to be administered as a vaccine contained the gp63gene. Also, the PSA-2 gene has been introduced into a plasmid and it hasbeen observed that it generates a Th-1 response and induction ofprotection. Vaccination with DNA plasmids which contain Ag-2 induce aTh-1 response and protect against infection with L. major, while Ag-2 instimulators immune complexes elicits a combined Th-1 and Th-2 responseand does not protect despite the fact that IFN-γ is induced. Equally,the gene encoding the LACK protein has been administered subcutaneouslyto BALB/c mice, in an expression vector which expresses the proteinunder the control of the cytomegalovirus promoter, and protectionagainst infection with L. major has been observed. In almost all casesin which DNA has been administered, the route has been intramuscular,although intradermal injection of particulate DNA must also be explored,as it requires a smaller amount of DNA . Other immunisation systems usevectors such as Salmonella, BCG or Vaccinia virus. It is interesting toremark that the inclusion of the gp63 gene in BCG is capable of inducingprotection against L. major.

The gene which encodes protein gp63 has also been introduced into genedelta araC under the control of the rac promotor in an attenuatedSalmonella typhimurium. Oral administration of 1×10⁹ colonies of thetransformed bacterium induces a T response within the scope of both Th-1and of cytotoxic cells against mastocytoma cells which express gp63.Protein gp63 in the form of gp63-ISCOMs complex induces protection inmice, evidenced by the reduction in inflammation and suppression oflesions. In serum, there are antibodies of the IgG2a type and,additionally, it is possible to observe a Th-1 response by induction ofIL-2, IFN-γ and IL-10. No DTH response was observed. Salmonella typhiNramp 1 transformed with gp63 elicits a Th-1 response with induction ofIL-2 and IFN-γ, and a strong resolution of the lesions is detected. Aprotein of L. pifanoi known as P-4 induces significant protectionagainst infection by Leishmania. Recent studies in humans with cutaneousleishmaniasis indicate that this protein or the peptides derived from itare capable of making T cells proliferate. There is no induction ofIL-4, whereas IFN-γ is induced. Aro-A and aro-D mutants of Salmonellatyphi transformed with IL-2, IFN-γ and TNF-α administered orally mayserve as therapeutic systems against infection by L. major. It isinteresting to observe that in these patients there is a greaterinduction of iNOS. The gp&3 gene has also been cloned in Aro-A and Aro-Dmutants, and it has been observed that after oral administration, theprotein encoded is capable of inducing significant protection againstinfection with L. major. This same protein is capable of inducingprotection when it is administered fused to several promoters inspecific varieties (GID105 and GID106) of Salmonella.

An artificial protein denominated Q has recently been described by ourgroup, which is composed of several antigenic fragments from 4 proteinsof Leishmania infantum (more specifically, Lip2a, Lip2b, PO and H2A),which, after being used as antigen,! has proven to have an importantvalue for the diagnosis of canine leishmaniasis, with a 93% sensitivityand a 100% specificity when compared with sera of control animals whichare not infected. Equally, our group has demonstrated that protein hsp70of Leishmania infantum is an important target of the immune response ininfections caused by infection with this parasite.

With the object of exploring the possibility that protein Q may be usedto design protection systems against infection by Leishmania infantum,both on its own as in combination with Hsp70, three series ofexperiments were designed using the hamster as a model. An experimentwas designed to check whether immunisation with Protein Q protected theanimals against infection on the short term, another to check whetherimmunisation protected them on the long term and the third was to checkthis protective effect after immunisation with the two proteinstogether. It was thereafter observed both from the short term analysisas well as from the long term analysis, that protein Q was capable ofeliciting an immune response which reduces the parasitic load both inthe Liver and in the Spleen after infection by Leishmania infantum inmost of the immunised animals, and that immunisation with the proteinsQ+Hsp70 also induced a significant response against both proteins andlead to a significant reduction of the parasitic load in most of theimmunised animals.

EXAMPLE 1 Immunisation with Protein Q

4 animals were immunised with 5 micrograms of protein Q dissolved in 40microliters of Freund's adjuvant, and another 4 animals were immunisedwith the same amount of adjuvant emulsified with 40 microliters of PBSsaline solution without the protein. In the first immunisation, Freund'scomplete adjuvant was employed combined with the protein, while in thetwo subsequent immunisations, incomplete Freund's adjuvant was usedmixed with the protein in the same proportion of protein/adjuvant. Threeintraperitoneal immunisations were carried out at 15 day intervals.Starting from the second week after each immunisation and throughout thewhole period of immunisation, blood samples were extracted to measurethe humoral response against protein Q in ELISA assays. It was observedthat already in the second week after the first immunisation there is apositive IgG response against protein Q, and that this response was highafter the second week after infection, and increased with time ofimmunisation reaching titres of 1/100.000. Equally, it was observed thatimmune response against protein Q was not modified significantly afterthe infection with the parasite FIG. 1.

Fifteen days after the third immunisation, the animals were infectedwith a dose of 10⁵ promastigote parasites, differentiated from infectiveamastigotes originating from an infected hamster. It had been previouslychecked that the inoculum was capable of inducing a strong parasitemiatogether with the disease four months after having administered theparasites, in 100% of the animals infected. Table 1 indicates the levelof parasitemia per mg of tissue both in liver and in the spleen of thecontrol and the vaccinated animals. It is possible to observe that inall of the vaccinated animals the parasitic load in the liver decreaseswith respect to the controls, and that this happens in a verysignificant manner in 75% of them. When the parasitic load in the spleenis examined, it possible to observe that also in 75% of the animals thisload was significantly lower than that of the controls, the RPL reaching83%-86%. The animal in which the RPL was 20% in the liver, had a 48% onein the spleen.

Table 1. Parasitic load in the liver and spleen of hamsters vaccinatedwith protein Q via the intraperitoneal route. After four weeks ofinfection, the parasitic load was measured by the method of limitdilutions(short term). The parasitic load is expressed as parasites permilligram of tissue. RPL reduction in parasitic load in t.

Hamster Immunized with the Q Protein

Hamster Liver (RPL) Spleen (RPL) 1 4 ± 1 (71%) 49 ± 3 (83%) 2 3 ± 2(78%) 39 ± 2 (86%) 3 5 ± 1 (64%) 50 ± 4 (83%) 4 11 ± 3  (20%) 153 ± 19(48%)

The numbers represent the mean of three determinations

Controls

4 hamsters 14 ± 5 295 ± 30

The number represent the mean of the 4 animals

EXAMPLE 2

In order to verify the effect of vaccination with protein Q on thereduction of the parasitic load on the long term, using another route ofinoculation, 4 animals were injected subcutaneously with 5 micrograms ofprotein Q dissolved in 40 microliters of PBS and mixed with 40microliters of Freund's adjuvant. In the first immunisation, Freund'scomplete adjuvant was employed, while in the two subsequent ones,incomplete Freund's adjuvant was used as indicated above. Vaccinationwas administered in three doses spaced at 15 day intervals. Fifteen daysafter the third immunisation they were administered an inoculum of 10⁵infective parasites. During all of the immunisation period andthroughout the whole of the infection (five months) blood was extractedto determine the kind of humoral response against protein Q and againstthe total proteins of the parasite. FIG. 2 shows that already after thesecond week of immunisation, the response against protein Q waspositive, as in the previous case, in three of the mice, and that theresponse against the protein was very high after two weeks of the firstimmunisation. The response kept on being very high after the remainingimmunisations reaching a titre of 1/75.000 on the week following thethird immunisation. In one of the animals the response against protein Qwas slower in relation to time, although the response reached the levelof that in the other animals by the end of the experiment. Consequently,from the data derived both from example 1 and from example 2, it ispossible to conclude that the degree of the immune response against theprotein, by both routes, intraperitoneal and subcutaneous, is veryrapid, although the response via the intraperitoneal route attainedhigher titres in the same times (1/75.000 versus 1/30.000). FIG. 3indicates the response against protein Q in the control animals. It ispossible to observe that reactivity against this protein is detected onthe 12-14th week after infection, which is when the first symptomsattributable to a potential leishmaniasis begin to be detected in theanimals infected. Table 2 shows the levels of parasitemia in the liverand spleen of the control and vaccinated animals. It can be seen that50% of the animals were protected at the level of the liver, in thesense that the reduction in the level of parasitemia was very high(87-89%), One of the animals was not protected, whereas in another ofthe animals the reduction of the parasitic load was of 22%. On thecontrary, the reduction of the parasitic load was of 98-99% in 100% ofthe animals at the level of the spleen.

Table 2. Parasitic load in the liver and spleen of hamsters vaccinatedwith protein Q by the subcutaneous route. After 20 weeks of infection,the parasitic load was measured by the method of limit dilutions (longterm). The parasitic load is expressed as parasites per milligram oftissue. RPL=reduction in parasitic load in %.

Hamsters Immunized with the Q Protein

Hamster Liver (RPL) Spleen (RPL) 1 1.4 × 10⁶ (22%) 1.0 × 10⁷   (98%) 22.0 × 10⁵ (89%) 4.9 × 10⁵ (99.9%) 3 2.4 × 10⁵ (87%) 3.3 × 10⁵ (99.9%) 42.4 × 10⁶  (0%) 6.3 × 10⁵ (99.9%)

The number represent the mean of three determinations

Controls

4 hamsters 1.8 × 10⁶ ± 1.2 × 10⁵ 5.2 × 10⁸ ± 2.6 × 10⁷

The numbers represent the mean of the parasitic load of the 4 animals

With the object of testing if protein Q could be used to designprotection systems in formulations which contained protein LiHsp70 ofLeishmania infantum, an experiment was carried out in Balb/c mice. Itwas observed that after immunisation, the parasitic load both in theliver as in the spleen was reduced significantly, being in some of theanimals, four orders of magnitude lower.

EXAMPLE 3 Immunisations with Protein Q+Protein Hsp70 in Freund'sAdjuvant

Each one of 4 hamsters were injected intraperitoneally with 5 microgramsof protein Q and 5 micrograms of protein LiHsp70 dissolved in 40microliters of PBS and emulsified in 40 microliters of Freund'sadjuvant. In the first immunisation, Freund's complete adjuvant wasemployed, while in the two subsequent ones, incomplete Freund's adjuvantwas used as indicated above. Vaccination was administered in three dosesspaced at 15 day intervals.

Fifteen days after the third immunisation they were administered aninoculum of 10⁶ infective parasites via the intracardiac route and weresacrificed at week 22. Every two weeks, for all of the immunisationperiod and throughout the whole of the infection, blood was extractedfrom them to determine the degree of humoral response against protein Qand against protein LiHsp70.

The relative parasitic load between the mice immunized with the Qprotein plus LiHsp70 is shown in Table 3. It was observed that all theanimals were highly protected and in some of them, in the spleen, thereduction was close to two-three orders of magnitude.

Table 3. Relative parasitic load of Balb/c mice immunized with the Qplus LiHsp70 protein after 4 month of infection.

Mice Immunized with the Protein Q and LiHsp70

Mouse Liver (RPL) Spleen (RPL) 1 1.5 × 10³ (97%) 1.5 × 10⁴ (99%) 2 0.9 ×10³ (98%) 1.4 × 10³ (99%) 3 2.0 × 10⁴ (60%) 5.6 × 10⁴ (99%) 4 0.8 × 10⁴(84%) 1.2 × 10⁵ (96%)

The numbers represent the mean of three determinations.

Controls

4 mice 5.1 × 10⁴ ± 2 × 10³ 3.2 × 10⁶ ± 8 × 10⁴

The numbers represent the mean of the parasitic load of the 4 animals.

Tables 4 and 5 shows that all the animals from Examples 1 and 2 respondimmunologically to protein Q, and that starting from the second weekafter immunisation the response against protein Q was high and thisresponse increased after the second immunisation (week 4). Table 6 showsthe response against protein LiHsp70 throughout the whole time of theexperiment was also positive, although lower than the response inExample 3 against protein Q.

Tables 4 and 5. Show the immune response in 4 hamsters injectedintraperitoneally with 5 micrograms of protein Q (TABLE 4—Example 1,short term) and the immune response in 4 hamsters injected issubcutaneously with 5 micrograms of protein Q (TABLE 5—Example 2, longterm).

TABLE 4 Example 1, short term Mice Immunized with the Q protein (Shortterm) Immune response against the Q protein Week Mouse 1 Mouse 2 Mouse 3Mouse 4  2 0, 8 0, 2 0, 3 0, 6  4 1, 9 2, 3 1, 1 2, 1  6 3 3 2, 4 2, 9Infected  8 3 3 2, 9 3 10 3 3 3 3 Control mice Immune response againstthe Q protein (Short term). Week Mouse 1 Mouse 2 Mouse 3 Mouse 4  2 0 00 0  4 0 0 0 0  6 0 0 0 0  8 0 0 0 0 10 0 0 0 0 The sera (diluted 1/200)showing an optical density of 3 have a titer higher than 1/45,000. Thesera were diluted 1/200

TABLE 5 Example 2, long term Mice immunized with the Q protein (Longterm). Immune response against the Q protein Week Mouse 1 Mouse 2 Mouse3 Mouse 4  2 1, 8 1, 3 1, 9 0, 4  4 2, 1 1, 8 2, 2 0, 7  6 2, 5 2, 7 2,5 0, 5  8 2, 8 2, 2 2, 2 0, 8 10 3 2, 9 2, 5 0, 7 12 3 3 3 0, 9 14 3 3 31 16 3 3 2, 9 1, 5 18 3 3 3 1, 7 20 3 3 3 2, 2 22 3 3 3 2, 6 24 3 3 3 326 3 3 3 Control mice Immune response against Q protein (Long term).Week Mouse 1 Mouse 2 Mouse 3 Mouse 4 2 0 0 0 0 4 0 0 0 0 6 0 0 0 0 8 0 00 0 10 0 0 0 0 12 0, 2 0, 1 0, 3 0, 1 14 0, 3 0, 5 0, 4 0, 2 16 0, 4 0,5 0, 7 0, 3 18 0, 9 1, 1 1, 7 0, 6 20 1, 1 1, 3 1, 8 0, 8 22 2, 5 2, 32, 7 1, 9 24 2, 3 2, 1 1, 8 2, 2 26 1, 9 2, 2 2, 3 2, 7 The sera(diluted 1/200) showing an optical density of 3 have a titer higher than1/45,000. The sera were diluted 1/200

TABLE 6. Shows the immune response in 4 hamsters injectedintraperitoneally with 5 micrograms of protein Q plus 5 micrograms ofprotein LiHSP 70 (Example 3, long term)

Mice immunized with the Q protein + Hsp70 Immune response against the Qprotein Week Mouse 1 Mouse 2 Mouse 3 Mouse 4  2 0, 4 0, 6 0, 5 0, 4  41, 6 1, 1 1, 6 1, 7  6 3 2 1, 9 1, 9  8 3 2, 4 2, 6 2, 6 10 3, 2, 9 3 2,9 12 2, 7 2, 9 2, 9 2, 9 14 2, 5 2, 6 2, 9 2, 7 16 2, 6 2, 5 2, 5 2, 418 2, 8 2, 7 2, 7 2, 5 20 2, 5 2, 8 2, 4 2, 7 22 2, 5 2, 4 2, 8 2, 9Control mice Immune response against the Q protein Week Mouse 1 Mouse 2Mouse 3 Mouse 4  2 0 0 0 0  4 0 0 0 0  6 0 0 0 0  8 0 0 0 0 10 0 0 0 012 0 0, 1 0, 2 0, 1 14 0, 3 0, 4 0, 2 0, 5 16 0, 5 0, 6 0, 7 0, 7 18 0,8 1, 4 1, 1 1, 5 20 1, 8 1, 6 1, 8 2, 2 The sera were diluted 1/800 Thesera were diluted 1/200

EXAMPLE 4 Immunization with Protein Q+BCG in Balb/c Mice

Four Balb/c mice were each injected intraperitoneally with 5 microgramsof protein Q and 10⁶ Colony Forming Units (CFU) of BCG (bacilleCalmette-Gu_rin) dissolved in 40 microliters of PBS. Immunization waseffected in three doses, 15 days apart. 15 days after the thirdimmunization, they were administered an inoculum of infective parasitesof 10⁵ BCN150 of L. infantum by the intracardiac route. Every two weeks,throughout the time of immunization and throughout infection, bloodsamples were taken from them for testing the degree of humoral responseto protein Q. The animals were sacrificed at week 8 after infection.

Table 7 (a and b) shows that the immunized animals respond positively toprotein Q starting from the second week and that the response increasesprogressively until in many cases it reaches values of 400,000. Table 8shows the difference in parasitic burden between the liver and spleen ofthe vaccinated and control animals.

Table 7: Immune response to protein Q in Balb/c mice immunized with 5micrograms of protein Q and 10⁶ CFU of BCG

Mice 1, 2, 3, 4 immunized

Mice 5, 6, 7, 8 unimmunized controls

TABLE 7 Reaction to protein Q week Mouse 1 Mouse 2 Mouse 3 Mouse 4preimmune 0 0 0 0 1st immuniz. 1 0 0.2 0.1 0 2nd immuniz. 3 0.5 0.7 0.80.9 3rd immuniz. 6 1.6 2.1 2.6 2.7 7 1.8 2.3 2.4 2.1 9 2.1 2.6 2.5 1.911 2.7 2.9 3 3 13 3 3 3 3 week Mouse 5 Mouse 6 Mouse 7 Mouse 8 preimmune0 0 0 0 1st immuniz. 1 0 0 0 0 2nd immuniz. 3 0 0 0 0 3rd immuniz. 6 0 00 0 7 0 0 0 0 9 0 0 0 0 11 0 0 0 0 13 0 0 0 0

the number 3 is equivalent to overflow in the measurement system

the sera were taken at the beginning of each of the the weeks indicatedthe 1st immunization was effected on day 0, the 2nd on day 15 and thethird on day 30.

Table 8. Parasitic burden in liver and spleen of Balb/c mice vaccinatedwith protein Q subcutaneously+10⁶ of BCG. 8 weeks after infection, theparasitic burden was measured by optical microscopy of tissueimpressions by measuring different fields containing 7000 nucleatedcells. The parasitic burden is represented as the quantity of parasitesper milligram of tissue, having previously calculated that 1 parasiteper 1000 nucleated cells is equivalent to an approximate quantity of 210parasites per milligram of tissue. RPB=reduction of parasitic burden, %.

Mouse Liver (RPB) Spleen (RPB) 1 72  (82%) 457 (89%) 2 40  (90%) 207(95%) 3 46  (88%) n.d. (100%)  4 n.d. (100%)  90 (98%)

Controls

4 animals. Mean 400 ± 15 4155 ± 30

n.d. parasites could not be detected in 5000 nucleates cells

12 1 412 PRT Artificial Sequence Description of Artificial SequenceProtein Q 1 Met Arg Gly Ser His His His His His His Thr Asp Pro His AlaSer 1 5 10 15 Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly IleGlu Gly 20 25 30 Arg Pro Leu Ala Thr Pro Arg Ser Ala Lys Lys Ala Val ArgLys Ser 35 40 45 Gly Ser Lys Ser Ala Lys Cys Gly Leu Ile Phe Pro Val GlyArg Val 50 55 60 Gly Gly Met Met Arg Arg Gly Gln Tyr Ala Arg Arg Ile GlyAla Ser 65 70 75 80 Gly Ala Pro Arg Ile Ser Glu Phe Ser Val Lys Ala AlaAla Gln Ser 85 90 95 Gly Lys Lys Arg Cys Arg Leu Asn Pro Arg Thr Val MetLeu Ala Ala 100 105 110 Arg His Asp Asp Asp Ile Gly Thr Leu Leu Lys AsnVal Thr Leu Ser 115 120 125 His Ser Gly Val Val Pro Asn Ile Ser Lys AlaMet Ala Lys Lys Lys 130 135 140 Gly Gly Lys Lys Gly Lys Ala Thr Pro SerAla Pro Glu Phe Gly Ser 145 150 155 160 Ser Arg Pro Met Ser Thr Lys TyrLeu Ala Ala Tyr Ala Leu Ala Ser 165 170 175 Leu Ser Lys Ala Ser Pro SerGln Ala Asp Val Glu Ala Ile Cys Lys 180 185 190 Ala Val His Ile Asp ValAsp Gln Ala Thr Leu Ala Phe Val Met Glu 195 200 205 Ser Val Thr Gly ArgAsp Val Ala Thr Leu Ile Ala Glu Gly Ala Ala 210 215 220 Lys Met Ser AlaMet Pro Ala Ala Ser Ser Gly Ala Ala Ala Gly Val 225 230 235 240 Thr AlaSer Ala Ala Gly Asp Ala Ala Pro Ala Ala Ala Ala Ala Lys 245 250 255 LysAsp Glu Pro Glu Glu Glu Ala Asp Asp Asp Met Gly Pro Ser Arg 260 265 270Val Asp Pro Met Gln Tyr Leu Ala Ala Tyr Ala Leu Val Ala Leu Ser 275 280285 Gly Lys Thr Pro Ser Lys Ala Asp Val Gln Ala Val Leu Lys Ala Ala 290295 300 Gly Val Ala Val Asp Ala Ser Arg Val Asp Ala Val Phe Gln Glu Val305 310 315 320 Glu Gly Lys Ser Phe Asp Ala Leu Val Ala Glu Gly Arg ThrLys Leu 325 330 335 Val Gly Ser Gly Ser Ala Ala Pro Ala Gly Ala Val SerThr Ala Gly 340 345 350 Ala Gly Ala Gly Ala Val Ala Glu Ala Lys Lys GluGlu Pro Glu Glu 355 360 365 Glu Glu Ala Asp Asp Asp Met Gly Pro Val AspLeu Gln Pro Ala Ala 370 375 380 Ala Ala Pro Ala Ala Pro Ser Ala Ala AlaLys Glu Glu Pro Glu Glu 385 390 395 400 Ser Asp Glu Asp Asp Phe Gly MetGly Gly Leu Phe 405 410 2 1436 DNA Artificial Sequence Description ofArtificial Sequence DNA encoding protein Q 2 atgagaggat ctcaccaccaccaccaccac acggatccgc atgcgagctc gaacaacaac 60 aacaataaca ataacaacaacctcgggatc gagggaaggc ctttagctac tcctcgcagc 120 gccaagaagg ccgtccgcaagagcggctcc aagtccgcga aatgtggtct gatcttcccg 180 gtgggccgcg tcggcgggatgatgcgccgc ggccagtacg ctcgccgcat cggtgcctct 240 ggcgccccca ggatttcagaattctccgtg aaggcggccg cgcagagcgg gaagaagcgg 300 tgccgcctga acccgcgcaccgtgatgctg gccgcgcgcc acgacgacga catcggcacg 360 cttctgaaga acgtgaccttgtctcacagc ggcgttgtgc cgaacatcag caaggcgatg 420 gcaaagaaga agggcggcaagaagggcaag gcgacaccga gcgcgcccga attcggatcc 480 tctagaccca tgtccaccaagtacctcgcc gcgtacgctc tggcctccct gagcaaggcg 540 tccccgtctc aggcggacgtggaggctatc tgcaaggccg tccacatcga cgtcgaccag 600 gccaccctcg cctttgtgatggagagcgtt acgggacgcg acgtggccac cctgatcgcg 660 gagggcgccg cgaagatgagcgcgatgccg gcggccagct ctggtgccgc tgctggcgtc 720 actgcttccg ctgcgggtgatgcggctccg gctgccgccg ccgcgaagaa ggacgagccc 780 gaggaggagg ccgacgacgacatgggcccc tctagagtcg accccatgca gtacctcgcc 840 gcgtacgccc tcgtggcgctgtctggcaag acgccgtcga aggcggacgt tcaggctgtc 900 ctgaaggccg ccggcgttgccgtggatgcc tcccgcgtgg atgccgtctt ccaggaggtg 960 gagggcaaga gcttcgatgcgctggtggcc gagggccgca cgaagctggt gggctctggc 1020 tctgccgctc ctgctggcgctgtctccact gctggtgccg gcgctggcgc ggtggccgag 1080 gcgaagaagg aggagcccgaggaggaggag gccgatgatg acatgggccc cgtcgacctg 1140 cagcccgccg ctgccgcgccggccgcccct agcgccgctg ccaaggagga gccggaggag 1200 agcgacgagg acgacttcggcatgggcggt ctcttctaag cgactcgcca tctcttagcc 1260 tccttgtggt gcgcttgaggtgctctcgct ctgcttctcc ttgcagtgtt ggctgactct 1320 ggcgggtatg tgccgtcgcattacacccac ctctcccacc cctttgccct acgcgctcgc 1380 atgcgcaatc cgtgaatcatcgagggaagt ctctctgggt ggcagtgggt aagctt 1436 3 383 PRT ArtificialSequence Description of Artificial Sequence Protein Q 3 Ile Glu Gly ArgPro Leu Ala Thr Pro Arg Ser Ala Lys Lys Ala Val 1 5 10 15 Arg Lys SerGly Ser Lys Ser Ala Lys Cys Gly Leu Ile Phe Pro Val 20 25 30 Gly Arg ValGly Gly Met Met Arg Arg Gly Gln Tyr Ala Arg Arg Ile 35 40 45 Gly Ala SerGly Ala Pro Arg Ile Ser Glu Phe Ser Val Lys Ala Ala 50 55 60 Ala Gln SerGly Lys Lys Arg Cys Arg Leu Asn Pro Arg Thr Val Met 65 70 75 80 Leu AlaAla Arg His Asp Asp Asp Ile Gly Thr Leu Leu Lys Asn Val 85 90 95 Thr LeuSer His Ser Gly Val Val Pro Asn Ile Ser Lys Ala Met Ala 100 105 110 LysLys Lys Gly Gly Lys Lys Gly Lys Ala Thr Pro Ser Ala Pro Glu 115 120 125Phe Gly Ser Ser Arg Pro Met Ser Thr Lys Tyr Leu Ala Ala Tyr Ala 130 135140 Leu Ala Ser Leu Ser Lys Ala Ser Pro Ser Gln Ala Asp Val Glu Ala 145150 155 160 Ile Cys Lys Ala Val His Ile Asp Val Asp Gln Ala Thr Leu AlaPhe 165 170 175 Val Met Glu Ser Val Thr Gly Arg Asp Val Ala Thr Leu IleAla Glu 180 185 190 Gly Ala Ala Lys Met Ser Ala Met Pro Ala Ala Ser SerGly Ala Ala 195 200 205 Ala Gly Val Thr Ala Ser Ala Ala Gly Asp Ala AlaPro Ala Ala Ala 210 215 220 Ala Ala Lys Lys Asp Glu Pro Glu Glu Glu AlaAsp Asp Asp Met Gly 225 230 235 240 Pro Ser Arg Val Asp Pro Met Gln TyrLeu Ala Ala Tyr Ala Leu Val 245 250 255 Ala Leu Ser Gly Lys Thr Pro SerLys Ala Asp Val Gln Ala Val Leu 260 265 270 Lys Ala Ala Gly Val Ala ValAsp Ala Ser Arg Val Asp Ala Val Phe 275 280 285 Gln Glu Val Glu Gly LysSer Phe Asp Ala Leu Val Ala Glu Gly Arg 290 295 300 Thr Lys Leu Val GlySer Gly Ser Ala Ala Pro Ala Gly Ala Val Ser 305 310 315 320 Thr Ala GlyAla Gly Ala Gly Ala Val Ala Glu Ala Lys Lys Glu Glu 325 330 335 Pro GluGlu Glu Glu Ala Asp Asp Asp Met Gly Pro Val Asp Leu Gln 340 345 350 ProAla Ala Ala Ala Pro Ala Ala Pro Ser Ala Ala Ala Lys Glu Glu 355 360 365Pro Glu Glu Ser Asp Glu Asp Asp Phe Gly Met Gly Gly Leu Phe 370 375 3804 27 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 4 cctttagcta ctcctcgcag cgccaag 27 5 27 DNA ArtificialSequence Description of Artificial Sequence oligonucleotide 5 cctgggggcgccagaggcac cgatgcg 27 6 26 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 6 gaattctccg taaggcggcc gcgcag 26 730 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 7 gaattcgggc gcgctcggtg tcgccttgcc 30 8 30 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide 8gtcgacccca tgcagtacct cgccgcgtac 30 9 30 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 9 gtcgacggggcccatgtcat catcggcctc 30 10 32 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 10 tctagacccg ccatgtcgtc gtcttcctcgcc 32 11 29 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 11 tctagagggg ccatgtcgtc gtcggcctc 29 12 30 DNAArtificial Sequence Description of Artificial Sequence oligonucleotide12 ctgcagcccg ccgctgccgc gccggccgcc 30

What is claimed is:
 1. A protein which generates an immune responseagainst visceral leishmaniosis, comprising the amino acid sequence ofSEQ ID NO:1.
 2. A composition, comprising a protein according toclaim
 1. 3. A composition, according to claim 2, wherein the compositionis a pharmaceutical composition.
 4. A pharmaceutical compositionaccording to claim 3, for the prevention and treatment, in humans andanimals, of visceral leishmaniosis.
 5. A pharmaceutical compositionaccording to claim 4, further comprising a physiological adjuvantsuitable for intraperitoneal, subcutaneous or intramuscularadministration.
 6. A pharmaceutical composition according to claim 5,which is a vaccine.
 7. A pharmaceutical composition according to claim4, further comprising the Hsp70 protein of Leishmania infantum.
 8. Apharmaceutical composition according to claim 7, further comprising aphysiological adjuvant suitable for intraperitoneal, subcutaneous orintramuscular administration.
 9. A pharmaceutical composition accordingto claim 8, wherein the composition is a vaccine.